Supplement figure legendsFigure S1 Expression of IFN-β mRNA by RIG-I and Riplet expression

Riplet, RIG-I, dRIG-I or TICAM-1 encoding plasmids were transfected into HEK293cells, and 24 hours after transfection, total RNA was extracted and endogenous IFN-β

mRNA was examined by RT-PCR.

Figure S2 Cytoplasmic localization of Riplet and RIG-I

HA-tagged and FLAG-tagged RIG-I were expressed in HeLa cells, and their

intracellular localizations were observed by confocal microscope with anti-FLAG or

anti-HA antibodies. Riplet and RIG-I are localized in the cytoplasm but not nucleus.

Figure S3 Knockdown of Riplet

HA-tagged Riplet (100 ng) and siRNA ( 10 pmol) were transfected into HEK293 cells

in 24-well plate, and 48 hours after transfection, cell lysate was prepared and analyzed

by SDS-PAGE. HA-tagged Riplet was abolished by siRNA for Riplet (Riplet siRNA or

Riplet si-1) but not control si or control si-1 (upper panel). Total protein was stained by

CBB (lower panel).

Figure S4

(A) HEK293FT cells were transfected with RIG-I dRD (100 ng), Riplet (100 ng) and

HA-tagged ubiquitin (300 ng) in 6-well plate, and after 24 hours, immunoprecipitation

was carried out with anti-FLAG antibody and the precipitates were analyzed by western

blotting.

(B) HEK293FT cells were transfected with RIG-IC (100 ng), Riplet (100 ng) and HA-

tagged K63R or K48R mutant ubiquitin, after 24 hours, immunoprecipiation was

carried out with anti-FLAG antibody and then the precipitates were analyzed by western

blotting.

(C) HEK393FT cells were transfected with FLAG-tagged RIG-IC, HA-tagged Riplet-

DN, HA-Ub and/or HA-tagged Riplet and then after 24 hours, immunoprecipitation was

carried out with anti-FLAG antibody. The immunoprecipitates was analyzed by western

blotting.

Figure S5

HEK293FT cells were transfected with dRIG-I, RIG-I RD, HA-tagged ubiquitin and/or

Riplet. After 24 hours, the cell lysate were prepared and immunoprecipitation was

carried out with anti-FLAG antibody. The immunoprecipitates were analyzed by

western blotting with anti-HA or FLAG antibodies.

Figure S6

(A) Schematic representations of RIG-I 3KA and 5KA mutants. The magnification of C-

terminal region of RIG-I mutants are shown in the lower panel. The lysine residues at

888, 907 and 909 were replaced with alanine residues in RIG-I 3KA mutant. RIG-I 5KA

mutant harbors five amino acids substitutions of alanine residues for the lysine residues,

849, 851, 888, 907 and 909.

(B) The amino acids sequence of the RIG-I C-terminal region of RIG-I. The mutated

lysine residues, 849, 851, 888, 907 and 909, are bolded and underlined.

SupplementsMaterials and Methods

Confocal microscope

HeLa cells were plated onto coverslips in a 24-well plate. After cells were

adhered onto the coverslips, cells were transfected with plasmid encoding FLAG-tagged

RIG-I and HA-tagged Riplet. After 24 hours incubation, cells were washed twice in

PBS and fixed with PBS containing 3% formaldehyde for 30 minutes at room

temperature. After the fixation, cells were washed with PBS and permialized with 0.2 %

Triton X-100 in PBS for 15 minutes. The cells were washed with PBS, and coated with

1 % BSA in PBS for 10 minutes at room temperature. Primary antibodies were diluted

500 times with 1 % BSA, and the cells were incubated with the diluted primary

antibody for one hour. After three times washing with 1 % BSA in PBS, the cells were

incubated with secondary antibodies, Alexa Fluor 488 rabbit and Alexa Fluor 594

mouse antibody, for 30 minutes at room temperature. The coverslips were washed three

times with 1 % BSA in PBS, and then mounted onto slideglasses using 2.3 % DABCO

in PBS. Imaging of the cells were carried out using Olympus Fluoview laser scanning

confocal microscopy.

RT-PCR to detect IFN-β mRNA

HEK293 cells were plated onto 24-well plate. Cells were transfected with the

pEF-BOS expression vector encoding dRIG-I, full-length RIG-I, Riplet and/or TICAM-

1 by FuGENE HD reagent. 24 hours after transfection, total RNA was extracted using

TRIZOL reagent. cDNA was generated by M-MLV-reverse transcriptase (Promega)

with random 9mer, and then subjected to PCR using Ex-Taq (TAKARA). The DNA

sequences of primers are IFN-beta forward: ATT GCC TCA AGG ACA GGA TG, IFN-

beta reverse: CTA TGG TCC AGG CAC AGT GA, GAPDH forward: CAC AGT CCA

TGC CAT CAC TG and GAPDH reverse: TAC TCC TTG GAG GCC ATG TG.

Preparation of HCV poly-U/UC RNA

The HCV genotype 1b poly-U/UC RNA (from 9421 to 9480, Accession number:

EU867431) was synthesized by T7 RNA polymerase in vitro. The template dsDNA

sequences were; Forward: TAA TAC GAC TCA CTA TAG GGT TCC CTT TTT TTT

TTT CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTC CTT TTT TTT TC,

Reverse: GAA AAA AAA AGG AGA AAA AAA AAA AAA AAA AAA AAA AAA

AAA AGA AAA AAA AAA AGG GAA CCC TAT AGT GAG TCG TAT TA. The

synthesized RNA was purified by TRIZOL reagent (Invitrogen).

1

2

Figure S3

Rip

let s

iRN

A

Con

trol s

iRN

A

Rip

let s

i-1

Con

trol s

i-1

(A)

UbRIG-IdRD

*

anti-HA anti-FLAG(Ub) RIG-I dRD

RIG-I dRD + + + +Riplet + - + -HA-Ub + + + +

(B)

IP:FLAG (RIG-IC)WB:HA (HA-Ub)

IP:FLAG (RIG-IC)WB:FLAG (RIG-IC)

Ub

RIG-IC

RIG-IC + + -Riplet + + - HA-Ub K48R K63R WT

(C)

Figure S4

WB: FLAG

WB: HA

WB: FLAGWB: HA

WB: HA

<WCE>

<IP: FLAG>

Riplet + - + - + -dRIG-I + + - - - -RIG-I RD - - + + - -HA-Ub + + + + + +

Ub-RIG-I

Riplet

dRIG-IRIG-I RD

RipletdRIG-IRIG-I RD

Fig. S5

888

XX X

XX X

907909

735 925

851

3KA

(A)

(B)

5KA

888 907 909849

1

XX

735 925

RIG-I 3KA

RIG-I 5KA